1. Field of the Invention
The present invention relates to liposomes, and more particularly multilamellar vesicles for entrapping a gene, a gene entrapping liposomal preparation, and a process for the manufacture of the liposomal preparation.
2. Related Arts
The liposomes are lipid vesicles, have similar structure as cell membranes in the living body, and can be prepared by suspending a polar lipid film in a solvent. The liposomes have been classified from morphological and structural view points into (1) multilamellar vesicles (MLV, liposomes with multilayers), (2) small unilamellar vesicles (SUV, small liposomes with a single layer), and (3) large unilamellar vesicles (LUV, large liposomes with a single layer).
The liposomes can entrap in inner space and membrane layer thereof various materials from low molecular substances to high molecular substances such as nucleic acid, protein or the like. Therefore, techniques utilizing the liposomes as a vehicle or carrier for introduction of the gene into a mammalian or vegetable cell have been developed, for instance, as shown in the following literatures.
a) P. F. Lurquin "Nucleic Acids Res.", Vol. 6, page 3773 (1979); PA1 b) R. Franco et al "Dev. Plant Biol.", Vol. 5, page 237 (1980); PA1 c) P. F. Lurquin et al "FEBS Lett.", Vol. 125, page 183 (1981); PA1 d) Jap. Pat. No. Sho 57 (year of 1982)--43688(A); PA1 e) P. L. Felgner et al "Proc. Natl. Acad. Sci. USA", Vol. 84, page 7413 (1987); PA1 f) P. Pinnaduwage et al "Biochim. Biophys. Acta", Vol. 985, page 33 (1989); PA1 g) R. Fraley et al "J. Biol. Chem.", Vol. 255, page 10431 (1980); PA1 h) Jap. Pat. No. Sho 55 (year of 1980)--118415(A); PA1 i) M. S. Ridder et al "Science", Vol. 215, page 166 (1982); PA1 j) C. Nicolau et al "Proc. Natl. Acad. Sci. USA", Vol. 80, page 1068 (1983); PA1 k) W. B. Rizzo et al "J. Gen. Virol.", Vol. 64, page 911 (1983); PA1 l) Jap. Pat. No. Sho 59 (year of 1984)--213392(A); PA1 m) T. Itani et al "Gene", Vol. 56, page 267 (1987); PA1 n) N. Ballas et al "Biochim. Biophys. Acta", Vol. 939, page 8 (1988); PA1 o) J. Szelei et al "Biochem. J.", Vol. 259, page 549 (1989); PA1 p) Jap. Pat. No. Sho 64 (year of 1989)--47381(A); and PA1 q) Jap. Pat. No. Hei 2 (year of 1990)--135092(A).
However, it has been reported that the MLV which are most easy in preparation may give to DNA a damage of nick or the like during a stage of operation for introducing the gene DNA into the liposomes (see said Literature a) and that the MLV are not suitable for entrapping DNA, since an entrapping or catching efficiency on high molecular substances is low [R. M. Straubinger and Papahajopoulos "Methods in Enzymol.", Vol. 101, page 512 (1983)]. The SUV are also not suitable for entrapping the gene and the like nucleic acid, since sonication for preparing the liposomes may give a damage to the nucleic acid, and inner space thereof is small. While, it has been considered that the LUV are suitable for entrapping the nucleic acid or the like, since inner space thereof is larger than that of said MLV and SUV, and an operation for preparing the same shall not give significant damage to DNA, but all of processes for preparing LUV, namely a reverse phase evaporation method, an ether injection method. Ca.sup.2+ fusion method and the like require quite complicate and troublesome operations and thus it cannot be said, at the present time, that such a process is suitable for an industrial large scale production.
It was actual situation that an entrapping efficiency of nucleic acid or the like with negative charge becomes low, when the membrane of liposomes has no charge or has been negatively charged, and that an expression efficiency of cells transformed with such gene entrapping liposomes cannot be made so higher. Therefore, such techniques have been developed that a positively charged lipid is added for constitutional lipids of the liposomes, or a surface active agent or the like is added for accelerating the entrapping of nucleic acid due to an electrostatic binding force to increase the entrapping efficiency. For instance, following reports have been issued. A primary amine of stearylamine is added for preparing liposomes to increase an entrapping efficiency of nucleic acid and to attain a relatively high resistibility to deoxyribonuclease, so that a gene can be introduced into Escherichia coli or a protoplast (see said Literatures b and c). Good nucleic acid entrapping efficiency can also be attained to show quite high introduction efficiency into cells, in comparison with widely accepted calcium phosphate precipitation method, when a quaternary amine which is more basic than the primary amine is employed (see said Literatures e, f and n).
The present inventors have also made apparent in Jap. Pat. No. Hei 2-135092(A) (said Literature q) that the gene entrapping efficiency has correlation with basicity of lipid with positive charge, and that addition of a quaternary amine for preparing liposomes is more effective than that of a secondary or tertiary amine on introduction or gene into cells.